This project aims to increase our understanding of inhibitory development in the auditory brainstem. Evidence suggests that the nominally inhibitory projection from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) uses the excitatory neurotransmitter glutamate during early postnatal life. This further suggests that glutamate could mediate inhibitory synaptic plasticity, if co-released with GABA/glycine at individual MNTB-LSO synapses. Experiments will use electrophysiological and immunohistochemical approaches to test the hypothesis that glutamate and GABA/glycine are co-released at individual GABA/glycinergic MNTB-LSO synapses. Whole-cell patch clamp recordings in brainstem slices, together with pharmacological and minimal stimulation techniques and paired-pulse analysis, will be used to test for corelease. Double and triple immunofluorescent labeling for vesicular transporters of glutamate and GABA/glycine, together with confocal microscopy and colocalization analysis, will be used to determine whether glutamate and GABA/glycine colocalize. This will indicate whether glutamate release could accompany GABA/glycine release at the same terminal or whether it defines a functionally distinct, hitherto undescribed, glutamatergic sub-population of MNTB neurons. Conclusions from these experiments will augment our understanding of the cellular events that govern auditory brainstem development. If the study points to a fundamental mechanistic role for glutamate release, results could potentially shed light on inhibitory development generally and advance our understanding of brain disorders caused by abnormal inhibition, such as epilepsy.